High-dose chemotherapy (HDC) in high-risk breast cancer is one of the possible
approaches how to improve therapeutic results, eventually, to overcome the incurability of
the disease. In recent randomized studies superiority of HDC to conventional therapy has
not been unambiguously established. Nevertheless, many oncologists, as well as, patients
are so convinced of HDC benefits, that they are not willing to take part in randomized
studies.
At an ASCO Annual Meeting (American Society of Clinical Oncology) in May 1999 in Atlanta
– preliminary results of five large randomized studies phase III were presented (2
studies on metastatic breast cancer and 3 studies on high-risk breast cancer with
more than 10 positive lymph nodes). The ASCO was informed of an investigation into serious
scientific misconduct in a clinical trial that was presented in a plenary
session of its Annual Meeting. The results of Dr. Bezwoda’s research were presented at
ASCO’s Meeting as one of four plenary papers on the investigational therapy and was the
only one to clearly indicate a survival benefit in the high-dose regimen.
Preliminary results presented there, however, did not confirm the original hypothesis of
the high efficacy of HDC. It is necessary to wait for definite results (within two or
three years, because enrollment of patients either has been finished or is being finished
just now) and several parameters may change. In view of hitherto results, some
investigators think that there is no need to continue in similar intensive studies. Still
some believe that different modifications of therapeutic regimens or new, less toxic drugs
should be tested which may lead to more effective and safer HDC.

Many unique features of chronic myelogenous leukemia (CML) make it as a model for
studying the development of leukemia in humans. Chronic myeloid leukemia is a disease
of the hematopoietic stem cell that progress in a multistep fashion. The biphasic or
triphasic clinical course of the disease exemplies the multistep process of tumor
progression from the indolent chronic phase to a more aggressive and terminal blast
crisis. CML was the first neoplastic disease shown to be associated with consistent
karyotypic abnormality now known as the Philadelphia (Ph) chromosome. The result of the
Philadelphia chromosome translocation t(9;22)(q34;q11) is the transposition of the c-abl
oncogene from chromosome 9 to chromosome 22, where it is fused with part of the bcr
gene. The translocation generates a new hybrid bcr-abl gene which plays
a crucial role in the pathogenesis of CML. Presently, CML is perhaps the best
understood cancer in humans and the model of oncogenesis mediated by the Ph chromosome
translocation is one of the best-characterized example of gene activation in leukemia.

The differential sensitivity of examined human ovarian carcinoma cell lines (CH1,
A-2780 and SKOV-3) to the IMPDH inhibitor, benzamide riboside (BR), was demonstrated with
the aid of MTT assay. Present data show that all three examined ovarian carcinoma cell
lines were sensitive to the cytotoxic effects of BR in the order of sensitivity CH1,
SKOV-3, A-2780, (IC50 = 2.8, 4.0 and 7.4 microM, respectively). Although
the IC50 of SKOV-3 cells was similar to that previously determined by others,
more than 20% of SKOV-3 cells remained viable in a plateau up to 40 microM BR
concentration. This relative resistance of SKOV-3 cells to BR corresponded to the absence
of BR–induced apoptosis in SKOV-3 cells, which together with clearly demonstrated
sensitivity of CH1 cells to BR-induced apoptosis, established by flow cytometry (presence
of nuclei with sub-G0 DNA content, Annexin V binding) and western blotting
(poly-ADP-ribosyl-polymerase (PARP) cleavage), further stressed the role of drug-induced
apoptosis in the overall drug-induced cytotoxicity.

Nucleoli in cells of the granulopoietic proliferating
compartment in patients suffering from the refractory anemia of the myelodysplastic
syndrome

Granulocytic precursors of the granulopoietic proliferating compartment (GPC) were
investigated in patients suffering from refractory anemia (RA) of myelodysplastic syndrome
(MDS) to provide an information on the number of nucleoli and incidence of main nucleolar
types in these cells stained with the simple cytochemical procedure for the demonstration
of RNA. The results demonstrated that the incidence of main nucleolar types in all stages
of the granulopoietic proliferating compartment in RA patients of MDS generally did not
differ in comparison with that of control patients without a disturbed
granulopoiesis. In contrast, the number of nucleoli expressed by the values of the
nucleolar coefficient in all stages of GPC in RA patients was significantly smaller than
in control persons. In addition, the values of the nucleolar coefficient of myeloblasts in
patients with RA of MDS were close to those in patients with acute myeloid leukemias.

Cytotoxic effects of the antiviremic drug ribavirin
(1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) were evaluated in vitro
measuring micronucleus formation and cell proliferation kinetics in whole blood cultures
employing cytokinesis block (CB) micronucleus test. The cells were exposed to ribavirin
doses ranging from 0.05–0.65 micromol/ml at three different incubation times. The
frequency of micronuclei in treated samples demonstrated relatively low ability of
ribavirin to induce micronuclei. However, the lowest concentration of ribavirin markedly
changed the frequency of mononucleated and multinucleated cells, particularly binucleated
ones, which declined significantly. The decline in the frequency of binucleate cells was
followed with accumulation of greater number of mononucleate cells. Decreased
proliferation potential of lymphocytes treated with ribavirin indicates that cells are
arrested prior to metaphase. The present investigation showed that ribavirin is capable to
induce a delay in cellular proliferation at all the doses assayed. The study
demonstrated that CB micronucleus assay is simple and rapid method that can be used to
assess toxic effect of drugs in vitro.

The expression of proliferating cell nuclear antigen (PCNA) in
leukemia cell lines HL-60 and K-562 at the light and electron microscope level

A. Grzanka, Z. Skok, A. Janiak, D.Grzanka

Department of Clinical Pathomorphology, University School of Medical Sciences, 85-094
Bydgoszcz, Poland, e-mail: grzanka@wsp.bydgoszc.pl;
Department of Biology and Environment Protection, Pedagogical University, Bydgoszcz,
Poland;
Department of Clinical Pathomorphology, University School of Medical Sciences, Bydgoszcz,
Poland;
University School of Medical Sciences, Bydgoszcz, Poland

PCNA antigen was localized at the light and electron microscopes level in two human
leukemia cell lines HL-60 and K-562. PCNA expression was used to discriminate cycling from
non-cycling cells. PCNA protein at the level of the light microscope was present in 70% of
the cell in HL-60 cell line and in 65% of the cells in K-562 line. Streptavidin
immuno-gold method was used for localization of PCNA expression at the ultrastructural
level. Positive staining for this protein was seen as granular pattern in the nucleus and
in the cytoplasm. In the nucleus the gold particles were seen to be associated with
heterochromatin and euchromatin of the leukemia cells. In cytoplasm it was found on the
endoplasmic reticulum and associated with ribosomes. Controls of the leukemia cells
incubation with normal mouse serum showed no labelling at the light and electron
microscope level.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic
ribonuclease has been proven to have important biological properties as aspermatogenic,
antitumor, embryotoxic and immunosuppressive activities. Recently we published preliminary
results concerning the ability of bovine seminal ribonuclease (BS RNase) to induce time
dependent apoptosis in Con-A stimulated human lymphocytes and in human tumor cells based
on DNA content and cell cycle analysis. In this study we bring more confirmative data
concerning the concentration dependent in vitro induction of apoptosis in
stimulated human lymphocytes and tumor cells of three human cell lines using the most
sensitive and specific cytometric method for at present apoptosis determination
– the indirect TUNEL. BS RNase 50 microg/ml was proven to induce 49.7, 54 and 68.1%
apoptosis in the cells of the ML-2 myeloid cell line and two neuroblastoma cell lines:
NB-1 and NB-2, respectively. In Con A-stimulated human lymphocytes, BS RNase also induced
apoptosis, eventhough not so pronounced as in human tumor cell lines. In all cultures the
induction of apoptosis was proportional to BS RNase concentration ranging from 2–50
microg/ml and correlated with proportional decrease in 3H-thymidine
incorporation into the newly synthesized DNA. Side by side with the ability of BS RNase to
suppress the growth of human tumors transplanted to nude mice, these biological properties
determine this enzyme as a promising agent with potential clinical application.

Chronic myelogenous leukemia (CML) is a malignant disease of hematopoietic stem
cell with a biphasic or triphasic clinical course and most often, with a fatal
outcome. Significant progress in improving outcome for patients with CML has been achieved
over past years. This can be attributed to marked improvement in therapeutic protocols and
increased use of bone marrow transplantation (BMT) which remains the most effective option
for long–term disease control of patient with CML. The residual leukemic activity in
patients after BMT remains a central clinical question. To effectively monitor
minimal residual disease leukemic activity after BMT, molecular genetic techniques are
currently utilized in conjunction with cytogenetic assays. Because the clinical
significance of detection minimal residual disease in CML remains to be determined, we
performed cytogenetic analysis and PCR amplification technique in 37 Ph+ CML patients. All
patients received transplants for CML in Bratislava between years 1992 and 1999. Our
results suggest that PCR positivity after transplant is of limited prognostic significance
for particular individuals and can be used to identified groups of individuals at elevated
risk of relapse.

Two p53 germline polymorphisms, a BstUI in exon 4 and a MspI
in intron 6 were studied using polymerase chain reaction (PCR) based methods in 50
patients with bladder cancer and 145 healthy controls. Increased frequencies of the BstUI
and MspI A2 alleles were found to be associated with statistically non-significant
(p = 0.2308 and p = 0.5959) but increased odd ratios for bladder cancer (OR
1.44, 95% CI 0.82–2.27 and OR 1.20, 95% CI 0.61–2.33). Statistically significant
difference between patients with bladder cancer and controls was found in the distribution
of MspI genotypes. There was a significantly lower proportion of the
heterozygous A1A2 genotype in all patients but not in controls (p = 0.0354). The
results of this study suggest that BstUI and MspI germ line polymorphisms of
the tumor suppressor gene p53 marginally modify the risk of bladder cancer.

Leptin is a nonglycosylated protein produced mostly by adipocytes. The role of
leptin in body weight regulation through its anorectic effect in hypothalamus is very well
known. Less known are other leptin effects such as the stimulation of hematopoesis and
some parts of immunity system. The role of leptin in the pathogenesis of some malignant
tumors is discussed. Only a little is known about bone marrow adipocyte leptin
production.
We examined leptin concentrations in the sera from peripheral blood and bone marrow, the
percentage of bone marrow fat, the degree of bone marrow infiltration, the body mass index
(BMI) in 42 patients with lymphoproliferative diseases. We found that bone marrow has
significantly lower leptin levels (6,6 ± 10,9 ng/ml) than peripheral blood (9,1 ± 11,5
ng/ml) (p < 0.0001). Bone marrow and peripheral blood leptin levels have
also a significant thin correlation (r = +0.91, p < 0.0001). Bone
marrow (r = +0.55, p < 0.0005) and peripheral blood (r = +0.52, p <
0.0005) leptin concentrations are significantly correlated to BMI. Blood serum leptin (r
= +0.46, p < 0.003) and bone marrow leptin (r = +0.40, p <
0.01) are related to the bone marrow fat percentage. In addition we found a negative
correlation of blood serum leptin (r = -0.59, p < 0.0001) and bone
marrow leptin (r = -0.42, p < 0.005) to bone marrow malignant
infiltration. When we divided the patients into groups with bone marrow infiltration more
than 10% and without or less than 10% infiltration, the first group had significantly
lower peripheral blood (p < 0.001) and bone marrow (p
< 0.02) leptin. We also confirmed a relation of bone marrow fat and
infiltration (r = +0.49, p < 0.001).
Our results suggest a relationship among leptin levels in blood or bone marrow and
bone marrow infiltration in lymphoproliferative diseases. This fact needs further
investigation and an evaluation of its application in clinical practice.

This paper deals with determination of potential carcinogenic and inhibitory activity
of 10 compounds isolated from the ethanolic extract of Lilium candidum L.performed
by DC polarography. The series of investigated compounds consisted of two spirostanol
saponins, two pyroline derivatives, jatropham and its glucoside, flavonol kaempferol,
2-fenylethyl-alpha-l-arabinopyranosyl-(1->6)-beta-d-glucopyranoside,
2-phenylethylpalmitate and methylsuccinic acid. Carcinogenic, resp. mutagenic activity
data for these compounds, with the exception of kaempferol, are not available in
scientific literature.
All tested compounds were reduced in N,N-dimethylformamide in one or two well defined
steps. They also formed a reversible complex with alpha-lipoic acid, a compound
serving as an indicator of carcinogenic activity. The marginal diffuse current values of
these complexes served as a criterion of a very low potential carcinogenicity.
The inhibitory activity of isolates were studied in the presence of
12-O-tetradecanoylphorbol-13-acetate, a specific tumor promoter for an epidermal
carcinogenesis, and in the presence of a polyaromatic substance 7,12-dimetylbenz(a)anthracene
known as a carcinogen. The spirostanol saponins possessed the highest inhibitory
activity (> 70%) and jatropham (66%). The inhibitory activity of jatropham glucoside
was significantly lower (49%). Practically zero inhibitory activity was measured for the
2-phenylethylpalmitate and methylsuccinic acid.

The aim of this study was to compare antiemetic efficacy of three serotonin
antagonists, granisetron, tropisetron and ondansetron, during conditioning for autologous
stem cell transplantation (ASCT).
Forty-five malignant lymphoma patients (mean age 38 years, M:F 30:15), undergoing the
highly emetogenic regimen BEAM prior to ASCT, were randomized to receive IV granisetron
(G) 3 mg once a day, IV tropisetron (T) 5 mg once a day, or IV
ondansetron (O) 8 mg twice daily, for six days. The treatment groups were comparable
with respect to age, sex and previous experience of nausea and/or vomiting. Nausea
and/or emesis control failure was defined as a nausea lasting >= 4 hours
and/or >= 3 episodes of vomiting/24 h, emesis control failure as >= 3
episodes of vomiting/24 h. Both the period of chemotherapy (6 days) and the whole period
of observation (10 days) were evaluated.
Nausea and/or emesis control failure occurred in 24% of patients during the period of
chemotherapy and in 51% of patients throughout the whole period of observation, while
emesis control failed in 2% and 27% of patients, respectively. The efficacy of three
serotonin antagonists was comparable during the chemotherapy period (5 patients with
nausea and/or emesis control failure in the granisetron group, 2 in the tropisetron
group and 4 in the ondansetron group, p = 0.40). When evaluating the
whole period of observation, the antiemetic response to G and T was
significantly better than to O, nausea and/or emesis control failure having occurred in
7 (47%) patients treated with G, 5 (33%) patients treated with T, and 12 (80%)
patients treated with O, p = 0.03. The results concerning emesis control
failures were similar, G 4 (27%), T 1 (7%), O 7 (47%), p =
0.04. Headache was the only frequent side effect of serotonin antagonists (30% incidence).
All three serotonin antagonists sufficiently controlled nausea and vomiting during
high-dose chemotherapy (BEAM) administration in 67–87% of patients. In comparison with
ondansetron, both tropisetron and granisetron proved to be more effective after ASCT, when
emetogenic factors other than chemotherapy alone participated.

Medullary thyroid carcinoma (MTC) – clinical and
molecular aspects on the basis of own experience

L. Pomorski, J. Bartkowiak, H. Pisarek, M. Bartos, J. Narębski

Clinic of Endocrinological and General Surgery, Medical University of Lodz, 93-513
Lodz, Poland;
Department of Experimental Endocrinology and Laboratory Investigations, Medical University
of Lodz, Lodz, Poland;
Department of Molecular Biology, Medical University of Lodz, Lodz, Poland

In our clinic 19 615 patients were operated over 25 years on for goiter. Malignant
thyroid neoplasms were found in 1049 (5.3%) patients including 875 (83.4%) women and 174
(16.6%) men. Sixty two adult patients (42 women and 20 men were operated on for medullary
thyroid carcinoma (MTC). Thyroid cancer was diagnosed in this group pre– or
intraoperatively in 44 (71%) patients and postoperatively, on histologic examination, in
18 (29%) patients. These patients were reoperated. Radical operations (total thyroidectomy
with regional lymph node removal) were conducted in 43 (69.3%) patients and palliative
ones in 19 (30.7%) patients. After MTC surgery, MEN 2A (MTC and an adrenal tumor) were
diagnosed by means of imaging techniques (USG, CT) in 6 (9.7%) patients. All adrenal
tumors were unilateral. Five of these patients were operated, and pheochromocytoma was
confirmed by histopathologic examination. Two years after the MTC operation, 1 women
was lost to follow-up. After a year, she was admitted to hospital for severe
hypertension and died of cerebral hemorrhagia. Pheochromocytoma was revealed by autopsy.
All patients were treated complementarily after the MTC operation. Different combinations
of teleradiotherapy, chemotherapy and substitutive doses of levothyroxine were used. Ten
(23.2%) of 43 patients operated radically were reoperated 1–3 years after the first
operation due to loco-regional tumor recurrence. Radical reoperations were performed in
4 patients, and palliative ones in 6.
Over a 0.5–23-year follow-up period, 26 (41.9%) patients died, including 20 of
cancer, and 6 of other reasons. Four out of 36 living patients have clinical or
biochemical symptoms of neoplastic disease. The follow-up period of MEN 2 patients
operated on ranged from 1 to 6 years. Up to now, no tumor in the second adrenal
gland has been diagnosed in any of these patients. Genetic (molecular) tests performed in
31 out of 36 living patients revealed mutations of RET gene in 4 (12.9%).